Hijacking Pseudomonas aeruginosa active transporters across the outer membrane: Challenges and opportunities for drug transport

Antimicrobial resistance is a serious public health threat worldwide. The emergence of multi-drug resistance bacteria challenges the development of novel antibiotics. Pseudomonas aeruginosa (P. aeruginosa) is an Gram-negative opportunistic pathogen that infects burn, wound and cystic fibrosis (CF) patients. P. aeruginosa is intrinsically resistant to many antibiotics, and further acquired resistance limits treatment options. The high level of resistance to antibiotics arises mainly from the tight control of P. aeruginosa over influx and efflux of molecules across its outer membrane. P. aeruginosa outer membrane includes a large number of different types of porins and efflux pumps that enable nutrient acquisition and antibiotic resistance. Among them, P. aeruginosa UCBPP-PA14 encodes 35 TonB-dependent transporters (TBDTs), which are defined as high-affinity active transporters and permit the transport of siderophores, heme, heavy metals and carbohydrates. Several studies showed high levels of TBDTs expression under iron deprivation and their importance for P. aeruginosa growth in vivo, but, except for the high-affinity siderophores and heme transporters, few have determined the contribution of single TBDT in vivo. In a Trojan horse approach, mimetics of essential substrates complexed to drugs, including siderophore-antibiotics conjugates and non-iron metalloporphyrins, are employed to induce the expression of associated TBDT(s) and to increase the antibiotic transport through TBDTs, hijacking the bacterial transport machinery. In spite of promising antibacterial activity in vitro, P. aeruginosa was able to rapidly develop resistance against the Trojan horse conjugates by facile inactivation of the TBDT involved in their transport. To circumvent this resistance mechanism, basic research on the processes associated with P. aeruginosa transport capabilities and substrate specificities in vivo is seriously needed to guide rational development of novel and effective therapeutics. In order to prevent facile inactivation of TBDT, we aim at identifying essential P. aeruginosa TBDTs that contribute to the bacterial fitness in vivo. We addressed this aim (i) by quantifying the abundance of TBDTs in vitro, in preclinical and human patients samples and (ii) by evaluating the relevance of TBDTs in vivo. We thus developed an ultrasensitive targeted proteomic approach to determine absolutely the abundance of TBDTs in vitro and in various hosts. Proteomic analyses revealed a clear disctinction between TBDTs expression in vitro and in vivo, suggesting that there is a urgent need for suitable in vitro medium that more faithfully reflects the in vivo reality. Expression data also highlighted a subset of TBDTs, including endogenous siderophore, heme, non-iron metal and some xenosiderophore transporters, that was highly abundant among the different in vivo conditions. Based on these data, we generated different mutants of the abundant TBDTs and evaluated them in competitive fitness assays in vivo. Overall, these data suggested that P. aeruginosa primarily used its high-affinity siderophore pyoverdine for iron scavenging, whereas uptake capabilities for its other endogenous siderophores, xenosiderophores, heme-associated substrates and possibly copper and zinc uptake were all dispensable for in vivo fitness in an intranasal model. In conclusion, implications of these results in the development of future compounds implementing the Trojan horse approach were discussed.